Nanoparticles Prepared Base On Casein And Study Of Its Potential Application

Micelles composed of amphiphilic copolymers have been explored as carriers for hydrophobic drugs in aqueous solution.Biopolymers are an interesting alternative to synthetic polymers because of their potential loading for both hydrophilic and hydrophobic drugs.In particular,fabricating polymeric carries based on biopolymers via green chemistry process is obviously desirable for the biomedical applications.Caseins are the predominant components in milk and they are a family of phosphorylated proteins.The four casein constituents,αs1-,αs2-,β-andκ-casein, exist in approximate proportions of 3:0.8:3:1 by weight in cow milk and their molecular weights are 19000-25000 Da.All of the four casein molecules in cow milk are amphiphilic proteins and have no defined structure.In food system,casein has many functions,such as emulsification,water binding,fat binding and texturization. These merits endow casein with ideal matrix to fabricate nano-materials for drug delivery.Maillard reaction is a natural,nontoxic reaction which happens during the processing,cooking,and storage of foods.Maillard reaction has been studied extensively which conjugates protein and polysaccharide by linking the reducing end of the polysaccharide to the amines in the protein(terminus and amino groups of lysine).The products of the Maillard reaction have seldom been used for drug delivery although many applications in other fields have been reported.This thesis focuses on the preparation of the nanoparticles based on casein via green processes and the characterizations as well as the potential applications of the nanoparticles.Two kinds of particles were prepared.One is micelles self-assembled by casein-graft-dextran copolymer,which was prepared through the Maillard reaction. As the core of the micelles is hydrophobic,β-carotene was used as a hydrophobic model compound to study encapsulation and release properties of the micelles.In addition,the Maillard reaction improves the solubility ofβ-casein in acidic solution. The emulsifying activity ofβ-casein-graft-dextran copolymer in acidic solution was studied.Another kind of particles isβ-casein-graft-dextran/lysozyme nanoparticles produced by gelation of lysozyme.The core of the nanoparticles was somewhat hydrophilic.Two kinds of charged derivatives of pyrene and aurichlorohydric acid were used as polar model compounds to study encapsulation properties of the nanoparticles.This thesis contains following five parts: The first part is the study of micellizaion of casein-graft-dextran copolymer.Two natural biomacromolecules,casein and dextran,are used to prepare casein-graft-dextran copolymer through the Amadori rearrangement of the Maillard reaction,a chemical-and solvent-free reaction.The copolymer has a reversible pH-sensitive property:micellization at the pI of casein with a casein core and dextran shell structure and dissociation when pH differs from the pI of casein.The micelles produced at pH 4.6 have a spherical shape and their size is dependent on the grafting degree of the copolymer and the molecular weight of dextran.The micelles are able to encapsulate hydrophobic compounds such as pyrene.The second part is the study of acidic solution properties ofβ-casein-graft-dextran copolymer.β-Casein-graft-dextran copolymer was prepared with the Amadori rearrangement of the Maillard reaction.Dynamic light scattering study shows that at pH range 4.0-5.0 where is close to the isoelectric point ofβ-casein,the copolymers form micelles which are spherical verified by atomic force microscopy imaging.The size and existent time of the micelles depend on the graft degree and the length of dextran side chains of the copolymers.The copolymers with higher hydrophilicity dissolved in molecular state show a better emulsifying ability at pH 4.6 and 8.0.The third part is the study of simultaneous nanoparticle formation and encapsulation driven by hydrophobic interaction of casein-graft-dextran andβ-carotene. Casein-graft-dextran copolymer is soluble in pH 7.0 aqueous solution in molecular state whereasβ-carotene is extremely insoluble.By the method of dialysis or evaporation then dispersing in water,50%ethanol solvent of the copolymer andβ-carotene mixture was changed to 100%aqueous solvent.During this process,the solubility of hydrophobic complex of casein andβ-carotene decreases whereas the solubility of dextran increases gradually,forming the particles with casein andβ-carotene core and dextran shell.The particles have spherical shape and their hydrodynamic diameter is about 200 nm at pH 7.0 solution.The particles can be stored in dried form.The aqueous dispersion of the particles is stable against dilution, pH change,ionic strength change,FeCl₃ oxidation,and long time storage.The encapsulatedβ-carotene can be released by pepsin or trypsin hydrolysis.These characters of the particles provide a possibility for practical applications of the particles to deliver unstable and hydrophobic nutrients and drugs.The fourth part is the study of self-assembly ofβ-casein and lysozyme.Two proteins,linearβ-casein and globular lysozyme,were used to fabricate nanoparticles using a green process.The two proteins formed polydisperse electrostatic complex micelles in the pH range of 3.0-12.0 at the molar ratio ofβ-casein to lysozyme 0.4.β-Casein/lysozyme nanoparticles formed after heating the micelle solution.After a heat treatment at 80℃which is above the denaturation temperature of lysozyme,lysozyme gelated andβ-casein was trapped in the nanoparticles.The nanoparticles have spherical shape and their sizes depend on the pH of the heat treatment,and the molar ratio ofβ-casein to lysozyme.There are moreβ-casein molecules located on the surface for the nanoparticles produced at pH 10.0,whereas more lysozyme on the surface for the nanoparticles produced at pH 5.0.The nanoparticles display amphoteric property:they carry net positive charges and negative charges at pH lower and higher than their zeroζ-potential,respectively.The nanoparticles are stable and relatively hydrophobic at pH around 5 and 10.The net charges on the surface stabilize the nanoparticles in the aqueous solution.The fifth part is the study of the encapsulation properties of nanoparticles selfassembled byβ-casein-graft-dextran copolymer and lysozyme.Dextran was grafted ontoβ-casein through the Maillard reaction and the nanoparticles that have dextran shell were prepared based on the interaction between lysozyme andβ-casein and gelation of lysozyme.The nanoparticle size is influenced by graft degree ofβ-casein-graft-dextran copolymer and the efficiency of the nanoparticle formation is influenced by molar ratio ofβ-casein to lysozyme.The nanoparticles have spherical shape and a somewhat hydrophilic core.The nanoparticle solution is stable against pH change, ionic strength change and long time storage.The nanoparticle can encapsulate anionic compounds and gold particles.